1. Field of the Invention
This invention relates to recombinant streptavidin proteins that bind biotin, to recombinant streptavidin proteins having an altered affinity for binding biotin and to methods utilizing recombinant streptavidin proteins for the detection and isolation of targets. The invention also relates to nucleic acids encoding recombinant streptavidin proteins and to recombinant cells which contain and express proteins encoded by these nucleic acids.
2. Description of the Background
Streptavidin, and its functional homolog avidin have been extensively used in biological and medical science due in large part to their ability to specifically bind biotin. Binding has a very high affinity of about 10.sup.15 M.sup.-1, and is one of the strongest known non-covalent interactions (N. M. Green, Methods Enzymol. 184:5-13,1990). This extraordinary affinity, coupled with the ability of biotin and its derivatives to be incorporated easily into various biological materials, endows streptavidin-biotin systems with great versatility.
Although avidin and streptavidin have almost the same high affinity for biotin, they are different in many other respects. The two proteins have different molecular weights, electrophoretic mobilities and overall amino acid composition. Avidin is a glycoprotein found in egg whites and the tissues of birds, reptiles and amphibia. Like streptavidin, avidin has almost the same high affinity for biotin and exists as a tetramer with a molecular weight of between about 67,000 to about 68,000 daltons. Avidin also has a high isoelectric point of between about 10 to about 10.5 and contains carbohydrates which cause it to bind non-specifically to biological materials including cell nuclei, nucleic acids and lectins. These non-specific interactions make avidin less suitable than streptavidin for many applications.
Biotin, also known as vitamin H or cis-hexahydro-2-oxo-1H-thieno-(3,4)-imidazole-4-pentanoic acid, is an essential vitamin found in every living cell including bacteria and yeast. In mammals, the tissues having the highest amounts of biotin are the liver, kidney and pancreas. Biotin levels also tend to be raised in tumors and tumor cells. In addition to cells, biotin can be isolated from secretions such as milk which has a fairly high biotin content. Biotin has a molecular weight of about 244 daltons, much lower than its binding partners avidin and streptavidin. Biotin is also an enzyme cofactor of pyruvate carboxylase, trans-carboxylase, acetyl-CoA-carboxylase and beta-methylcrotonyl-CoA carboxylase which together carboxylate a wide variety of substrates.
Only the intact bicyclic ring of biotin is required for the strong binding to streptavidin. The carboxyl group of biotin's pentanoic acid side chain has little to contribute to this interaction. Consequently, biotin derivatives, reactive to a variety of functional groups, can be prepared by modifying the pentanoic acid carboxyl group without significantly altering the target's physical characteristics or biological activity. This allows biotin to be conjugated to a number of target molecules.
Streptavidin is produced by the bacteria, Streptomyces avidini, and exists as a tetrameric protein having four identical subunits. The full length streptavidin monomer is 159 amino acids in length, some 30 residues longer than avidin. It contains no carbohydrate and has an acidic isoelectric point of about 5.0 which accounts, in part, for the low non-specific binding level. Each subunit of streptavidin is initially synthesized as a precursor of 18,000 daltons which forms a tetramer of about 75,000 daltons. Secretion and post-secretory processing results in mature subunits having an apparent size of 14,000 daltons. Processing occurs at both the amino and carboxyl termini to produce a core protein of about 13,500 daltons, having about 125 to 127 amino acids. This core streptavidin forms tetramers and binds to biotin as efficiently as natural streptavidin. The amino acid sequence of the mature 160 amino acid protein is as follows:
1 XPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALT (SEQ ID NO 1) 41 GTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWK 81 NNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAW 121 KSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ-159
A natural streptavidin tetramer is formed by interdigitating a pair of streptavidin dimers with their dyad axes coincident (W. A. Hendrickson et al., Proc. Natl. Acad. Sci. USA 86:2190-94,1989). A tetramer is stabilized by numerous van der Waals forces, with subunits forming a symmetric dimer additionally connected by hydrogen bonds near the carboxyl terninus. This force distribution within a tetramer indicates that there are two classes of subunit interfaces. One interface is between subunits in a stable symmetric dimer and the other is between two stable dimers. When the dissociation of a tetramer occurs, it is likely that the interface between two stable dimers would be first disrupted because this has lower stability than that between two subunits in a stable dimer. If such dissociation occurred, the resulting dimeric molecules should have much reduced affinity for biotin because of the lack of contacts made by Trp-120 of an adjacent subunit to biotin through the dimer--dimer interface (A. Chilkoti et al., Proc. Natl. Acad. Sci. USA 92:1754-58,1995; T. Sano et al., Proc. Natl. Acad. Sci. USA 92:3180-84,1995). This might explain why the dissociation of biotin from streptavidin can be observed even under relatively mild conditions, despite the very high biotin-binding affinity of streptavidin.
The mature streptavidin tetramer binds one molecule of biotin per subunit and the complex, once formed, is unaffected by most extremes of Ph, organic solvents and denaturing conditions. Separation of streptavidin from biotin requires harsh conditions, such as 8 M guanidine, pH 1.5, or autoclaving at 121.degree. C. for 10 minutes.
The advantages of streptavidin-biotin binding systems are numerous. The exceptionally high affinity and stability of the complex ensures complete reaction. Biotin's small size allows it to be conjugated to most molecules with no loss in molecular activity. Multiplicity of biotinylation sites combined with the tetrameric structure of streptavidin allows for amplification of the desired signal. The system is extremely versatile, as demonstrated by the large number of functional targets, binders and probes. The system is amenable to multiple labelling techniques, a wide variety of biotinylated agents and streptavidin-containing probes are commercially available.
Streptavidin-biotin complexes are used in a number of diagnostic and purification technologies. In general, a target molecule to be purified or detected is bound either directly to biotin or to a biotinylated intermediate. The binder may be almost any molecule or macromolecule that will complex with or conjugate to a target molecule. For example, if a particular antigen is the target, its binder would be an antibody. The biotinylated target is bound to streptavidin which may be bound to a probe for ease of detection. This basic technique is utilized in chromatography, cytochemistry, histochemistry, pathological probing, immunoassays, bioaffmity sensors and cross-linking agents, as well as more specific techniques such as targeting, drug delivery, flow cytometry and cytological probing.
The origins of the unusually high binding affinity seen in streptavidin-biotin complexes has not been fully elucidated. X-ray crystallographic studies have shown that streptavidin's carboxyl and amino termini lie on the molecule's surface (P. C. Weber et al., J. Am. Chem. Soc. 114:3197-200, 1992). These termini have been modified by cleavage or conjugation with a minimal effect on biotin binding affinity.
The streptavidin-biotin complex does not involve any covalent bonds, but does contain many hydrogen bonds, hydrophobic interactions and van der Waal interactions. These interactions are largely mediated by the aromatic side chains of tryptophan. Two tryptophan-lysine pairs are conserved between streptavidin and avidin. These pairs are found at positions 79-80 and 120-121 in streptavidin. Additional tryptophan residues in streptavidin are found at positions 92, 108 and 120.
Although participation of tryptophan residues in biotin-binding has been indicated, a quantitative understanding of Trp-120's contribution to biotin-binding has not been reported. Streptavidin's six tryptophan residues per subunit make conventional chemical modifications of any one specific tryptophan residue difficult a situation exacerbated by the tetrameric nature of streptavidin. The Trp-120 of one particular streptavidin subunit makes contact with the biotin bound to an adjacent subunit (A. Pahler et al.,J. Biol. Chem. 262:13,933-37,1987). This residue contacts the alkyl moiety of biotin's pentanoyl group in an apparent hydrophobic interaction. Streptavidin's subunit association is made tighter upon biotin-binding (Advances in Biomagnetic Separation, T. Sano et al., Eaton Publishing, Natick, Mass., 1994). Because the contact made by Trp-120 to the biotin of an adjacent subunit occurs through the dimer--dimer interface, this residue possibly plays a key role in the biotin-induced tighter association of streptavidin. However, because streptavidin's Trp-120 residue is adjacent to a lysine, both of which are conserved in avidin, lysine may also have a role in binding. Lysine is known to play a critical role in avidin-biotin complex formation. For example, when an avidin lysine at positions 45, 94 or 111 is bound to a dinitrophenyl group, activity is abolished (Avidin-Biotin Chemistry: A Handbook, M. D. Savage et al., editors, page 7, 1992).
Trp-120 may play a role in maintaining local structures of streptavidin, particularly around the biotin-binding sites and the dimer--dimer interface. Strong hydrophobicity is observed around Trp-120 and three other tryptophan residues (Trp-79, 92 and 108) that make contact with biotin (P. C. Weber et al., Sci. 243:85-88,1989; C. E. Argara na et al., Nuc. Acids Res. 14:1871-82, 1986). In addition, hydrophobic interactions are the major force for the stable association of the two symmetric streptavidin dimers. Changes in local environment caused by the mutation of Trp-120 could prevent the molecule from folding correctly, resulting in diminished biotin-binding ability. In fact, the conversion of some amino acid residues located around the dimer--dimer interface to hydrophilic amino acids causes the formation of insoluble aggregates, probably due to random inter-molecular interactions.
Streptavidin's herculean affinity for biotin is unfortunately its major drawback. The streptavidin-biotin binding system is essentially irreversible. The streptavidin-biotin bond is not affected by pH values between 2 to 13, nor by guanidine-HCl concentrations up to 8 M (neutral pH). The half-life for spontaneous dissociation of the streptavidin-biotin bond is about 2.5 years. The extremely strong binding of biotin to streptavidin means that biotinylated proteins can only be recovered from streptavidin supports under denaturing conditions. This sort of system is inappropriate for many procedures such as, one of its principal uses, the purification of delicate proteins. Streptavidin-biotin cannot be used in sequential assays to detect specific types of biomolecules, macromolecular complexes, viruses or cells present in a single sample. The high affinity necessitates the use of harsh chemical reagents, complex procedures, and careful monitoring of the reactions. This also limits both yields and the ability to fully automate such reactions.
A number of methods have been developed in an attempt to create a releasible streptavidin-biotin or avidin-biotin conjugate. These methods include partly monomeric avidin beads, N-hydroxysuccinimide-iminobiotin and biotin or streptavidin cleavage.
Monomeric avidin beads are formed by denaturing tetrameric avidin and coupling the denatured protein to chromatography beads. Thus, the so-called monomeric avidin is really a mixture of monomeric, dimeric and tetrameric proteins that have a binding affinity distributed between the wild type affinity of 10.sup.15 M.sup.-1 and the reduced affinity of 10.sup.8 M.sup.-1. Thus, monomeric avidin beads produce low yields because some of the biotinylated products are irreversibly bound. Furthermore, the density and capacity of monomeric avidin beads is low.
N-hydroxysuccinimide-iminobiotin (NHS-iminobiotin) is a guanido analog of NHS-biotin with a pH sensitive binding affinity for streptavidin. The complete dissociation of NHS-iminobiotin from streptavidin occurs at low pH without the need for strong denaturants. The drawback to the NHS-iminobiotin system is that binding requires a pH of 9.5 or greater, while release requires a pH of less than 4. Thus, the use of NHS-iminobiotin is limited to those few molecules which are stable over a wide pH range.
One method used to dissociate the streptavidin-biotin bond involves proteinase K digestion of streptavidin (M. Wilchek et al., Anal. Biochem. 171:1-32, 1988). However, significant amounts of the streptavidin molecules remain attached even after proteinase K treatment. Proteinase K is useful only when the biotinylated product does not comprise proteins. Furthermore, this system precludes sequential assays or transfers of target.
Another method of release involves biotin cleavage of the binding partners, for example, of a cleavable biotin such as immunopure NHS-SS-biotin which is commercially available (Pierce Chemical Co.; Rockford, Ill.). NHS-SS-biotin consists of a biotin molecule linked through a disulfide bond and an N-hydroxysuccinimide ester group that reacts selectively with primary amines. Using this group, NHS-SS-biotin is linked to a target molecule and the biotin portion removed by thiol cleavage. This complex approach is slow and of limited use since thiols normally disrupt native protein disulfide bonds. Furthermore, cleavage leaves a reactive sulfhydryl group that tends to react with other components of the mixture. Also, thiol-contaning nucleic acids will no longer hybridize, severely limiting their usefulness.